Method for measuring anti-wt1 antibody

ABSTRACT

Provided is an invention relating to a method for measuring an anti-WT1 antibody in a sample allowing measurement and evaluation of the anti-WT1 antibody with higher accuracy and to use of the method. The method for measuring an anti-WT1 antibody in a sample includes using a polypeptide having antigenicity to the anti-WT1 antibody selected from a polypeptide comprising the amino acid sequence of positions 294-449 in SEQ ID NO: 1, a partial polypeptide of the polypeptide, and a polypeptide including an amino acid sequence having deletion, substitution, or addition of one to several amino acids in the amino acid sequence constituting each of these polypeptides and/or a polypeptide having antigenicity to the anti-WT1 antibody selected from a polypeptide comprising the amino acid sequence of positions 181-324 in SEQ ID NO: 1, a partial polypeptide of the polypeptide, and a polypeptide including an amino acid sequence having deletion, substitution, or addition of one to several amino acids in the amino acid sequence constituting each of these polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.14/239,449, filed Feb. 18, 2014, which is a continuation ofInternational Application No. PCT/JP2012/073512, filed Sep. 13, 2012which is based upon and claims the benefits of priority to JapaneseApplication No. 2011-200620, filed Sep. 14, 2011. The entire contents ofall of the above applications are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method for measuring an anti-WT1antibody in a sample using a WT1 fragment.

BACKGROUND ART

A WT1 gene (Wilms tumor gene) is a zinc finger transcription factorisolated as a responsible gene for Wilms tumor. Abnormally highexpression of the WT1 gene was then confirmed in acute myeloid leukemiaand also in various solid cancers (Non Patent Documents 1 to 3), andapplication of a WT1 protein as a peptide vaccine has been tried.

In recent years, it has been revealed that the WT1 protein has astructure including a repression domain, an activation domain, and azinc finger domain, which is a DNA-binding domain, and regulates geneexpression by binding to an early growth response protein 1 (EGR-1)region. A function as a tumor suppressor gene has also been reported(Non Patent Documents 4 and 5).

In addition, the presence of autoantibodies to the WT1 protein has beenrevealed. It has been reported that the titer of autoantibody againstthe WT1 protein is particularly high in blood of hematological cancer orlung cancer (small cell cancer) patients (Non Patent Documents 6 to 8).Higher expression of WT1 mRNA tends to cause poor prognosis. Incontrast, it has been reported that a higher blood level of an anti-WT1antibody tends to cause good prognosis (Non Patent Document 7).Accordingly, it is believed that accurate measurement of the anti-WT1antibody in patient blood is useful for selection of a method oftreatment or monitoring of treatment progress. For example, measurementof an anti-WT1 antibody using a WT1 protein containing the repressiondomain and the activation domain but lacking the zinc finger as anantigen has been reported (Patent Documents 1 and 2).

However, the mechanism by which an in vivo protein becomes to berecognized as a foreign substance and induces production of anautoantibody thereto is still unclear. In addition, the concentration ofthe antibody is very low, and a method for detecting an autoantibodywith high sensitivity has not been established. Also regarding theanti-WT1 antibody, there is a problem that the known method using a WT1protein antigen cannot necessarily accurately evaluate the antibodybecause of the narrow titer distribution of the detected antibody.

CITATION LIST Patent Document

[Patent Document 1] JP-A-2002-48793

[Patent Document 2] JP-A-2006-267124

Non Patent Document

[Non Patent Document 1] Inoue K, Sugiyama H, Ogawa H, et al., WT1 as anew prognostic factor and new marker for the detection of minimalresidual disease in acute leukemia, Blood 1994, 84: 3071-9

[Non Patent Document 2] Oji Y, Miyoshi S, Maeda H, et al.,Overexpression of the Wilms' tumor gene WT1 in de novo lung cancers, IntJ Cancer 2002, 100: 297-303

[Non Patent Document 3] Miyoshi Y, Ando A, Egawa C, et al., Highexpression of Wilms' tumor suppressor gene predicts poor prognosis inbreast cancer patients, Clin Cancer Res 2002, 8: 1167-71

[Non Patent Document 4] Haber D A, Sohn R L, Buckler A J, et al.,Alternative splicing and genomic structure of the Wilms' tumor gene WT1,Proc Natl Acad Sci USA 1991, 88: 9618

[Non Patent Document 5] Madden S L, Cook D M, Morris J F, et al.,Transcriptional repression mediated by the WT1 Wilms tumor gene product,Science 1991, 253: 1550

[Non Patent Document 6] Olga AEl, Oka Y, Tsuboi A, et al., Humoralimmune responses against Wilms tumor gene WT1 product in patients withhematopoietic malignancies, Blood 2002, 99: 3272-3279

[Non Patent Document 7] Oji Y, Kitamura Y, Kamino K, et al., WT1 IgGantibody for early detection of nonsmall cell lung cancer and as itsprognostic factor, Int J Cancer 2009, 125: 381-7

[Non Patent Document 8] Tamura H, Dan K, Yokose N, et al., Prognosticsignificance of WT1 mRNA and anti-WT1 antibody levels in peripheralblood in patients with myelodysplastic syndromes, Leukemia Res 2010, 34:986-990

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

It is an object of the present invention to provide a method formeasuring an anti-WT1 antibody that enables more accurate measurementand evaluation of the anti-WT1 antibody in a WT1-associated diseasepatient and use of the method.

Means for Solving the Problems

The present inventors have investigated detection of an anti-WT1antibody in blood of WT1-associated disease patients and have found thatan in vivo autoantibody to an antigen is produced when the antigenrecognized by the autoantibody is conformationally modified to expose anepitope site to the surface and is thereby recognized as a foreignsubstance. The inventors have also found that the major epitopes of WT1are the central region (amino acid numbers: 181-324) and the C-terminalregion (amino acid numbers: 294-449) of the amino acid sequence (SEQ IDNO: 1) constituting the human WT1 protein and that the anti-WT1 antibodytiter in measurement of the anti-WT1 antibody using a polypeptidefragment corresponding to the C-terminal region (zinc finger region), aDNA biding domain of WT1, as an antigen particularly shows a highcorrelation with that in the measurement using the full-length WT1 as anantigen. The inventors further have found that in comparison between thetiter distribution of antibodies in healthy subjects and those in cancerpatients, the titer distribution of antibodies in the cancer patients isbroader than that in the healthy subjects to show a significantdifference, which allows accurate and sensitive determination of a hightiter group of the anti-WT1 antibody, such as cancer patients.

Accordingly, the present invention relates to the following aspects 1)to 9):

1) A method for measuring an anti-WT1 antibody in a sample, the methodcomprising using a polypeptide having antigenicity to the anti-WT1antibody selected from a polypeptide comprising the amino acid sequenceof positions 294-449 in SEQ ID NO: 1, a partial polypeptide of thepolypeptide, and a polypeptide including an amino acid sequence havingdeletion, substitution, or addition of one to several amino acids in theamino acid sequence constituting each of these polypeptides and/or apolypeptide having antigenicity to the anti-WT1 antibody selected from apolypeptide comprising the amino acid sequence of positions 181-324 inSEQ ID NO: 1, a partial polypeptide of the polypeptide, and apolypeptide including an amino acid sequence having deletion,substitution, or addition of one to several amino acids in the aminoacid sequence constituting each of these polypeptides;

2) The method according to aspect 1), wherein a concentration of theantibody is measured by immobilizing at least one of the polypeptides toa solid phase and detecting a reaction product between the immobilizedpolypeptide or polypeptides and an anti-WT1 antibody present in asample;

3) A method for diagnosing a WT1-associated disease, the methodcomprising using a polypeptide having antigenicity to an anti-WT1antibody selected from a polypeptide comprising the amino acid sequenceof positions 294-449 in SEQ ID NO: 1, a partial polypeptide of thepolypeptide, and a polypeptide including an amino acid sequence havingdeletion, substitution, or addition of one to several amino acids in theamino acid sequence constituting each of these polypeptides and/or apolypeptide having antigenicity to the anti-WT1 antibody selected from apolypeptide comprising the amino acid sequence of positions 181-324 inSEQ ID NO: 1, a partial polypeptide of the polypeptide, and apolypeptide including an amino acid sequence having deletion,substitution, or addition of one to several amino acids in the aminoacid sequence constituting each of these polypeptides;

4) The method according to aspect 3), which determines prognosis ofleukemia;

5) A method for predicting a responder to or for therapeutic monitoringof WT1 vaccine therapy of cancer, the method comprising using apolypeptide having antigenicity to an anti-WT1 antibody selected from apolypeptide comprising the amino acid sequence of positions 294-449 inSEQ ID NO: 1, a partial polypeptide of the polypeptide, and apolypeptide including an amino acid sequence having deletion,substitution, or addition of one to several amino acids in the aminoacid sequence constituting each of these polypeptides and/or apolypeptide having antigenicity to the anti-WT1 antibody selected from apolypeptide comprising the amino acid sequence of positions 181-324 inSEQ ID NO: 1, a partial polypeptide of the polypeptide, and apolypeptide including an amino acid sequence having deletion,substitution, or addition of one to several amino acids in the aminoacid sequence constituting each of these polypeptides;

6) The method according to aspect 5), wherein the cancer is brain tumoror colon cancer;

7) A reagent for measuring an anti-WT1 antibody in a sample, the reagentcomprising a polypeptide having antigenicity to an anti-WT1 antibodyselected from a polypeptide comprising the amino acid sequence ofpositions 294-449 in SEQ ID NO: 1, a partial polypeptide of thepolypeptide, and a polypeptide including an amino acid sequence havingdeletion, substitution, or addition of one to several amino acids in theamino acid sequence constituting each of these polypeptides and/or apolypeptide having antigenicity to the anti-WT1 antibody selected from apolypeptide comprising the amino acid sequence of positions 181-324 inSEQ ID NO: 1, a partial polypeptide of the polypeptide, and apolypeptide including an amino acid sequence having deletion,substitution, or addition of one to several amino acids in the aminoacid sequence constituting each of these polypeptides;

8) A reagent for examining a WT1-associated disease, the reagentcomprising a polypeptide having antigenicity to an anti-WT1 antibodyselected from a polypeptide comprising the amino acid sequence ofpositions 294-449 in SEQ ID NO: 1, a partial polypeptide of thepolypeptide, and a polypeptide including an amino acid sequence havingdeletion, substitution, or addition of one to several amino acids in theamino acid sequence constituting each of these polypeptides and/or apolypeptide having antigenicity to the anti-WT1 antibody selected from apolypeptide comprising the amino acid sequence of positions 181-324 inSEQ ID NO: 1, a partial polypeptide of the polypeptide, and apolypeptide including an amino acid sequence having deletion,substitution, or addition of one to several amino acids in the aminoacid sequence constituting each of these polypeptides; and

9) A method for detecting an autoantibody, the method comprisingmodifying an in vivo protein such that an epitope site is exposed to thesurface and using the modified protein as an antigen recognized by theautoantibody.

Effects of the Invention

The method for measuring an anti-WT1 antibody of the present inventioncan accurately and sensitively measure the anti-WT1 antibody inWT1-associated disease patients and therefore allows satisfactorydetection of a variation in the anti-WT1 antibody titer. Accordingly, itis possible to easily and highly sensitively perform, for example,diagnosis of WT1-associated diseases, monitoring of therapeutic effects,determination of prognosis, prediction of a responder before vaccinetherapy, and monitoring of response to vaccine therapy after thetherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the titers of antibody against a full-lengthWT1 antigen in blood of cancer patients.

FIG. 2 includes graphs showing comparison between the titer of antibodyagainst the full-length WT1 antigen and the titers of antibody againstpartial WT1 antigens, i.e., the results of comparison of the titer ofantibody against the full-length WT1 antigen to the titer of antibodyagainst a Fr. 1 antigen (a), a Fr. 2 antigen (b), and a Fr. 3 antigen(c), with the vertical axis showing the titer (unit) of antibody againsta partial WT1 antigen and the horizontal axis showing the titer (WRU) ofantibody against a full-length WT1 antigen.

FIG. 3 is a graph showing comparison between the titer of antibodyagainst the Fr. 2 antigen and the titer of antibody against the Fr. 3antigen, with the vertical axis showing the titer (unit) of antibodyagainst the Fr. 3 antigen and the horizontal axis showing the titer(WRU) of antibody against the Fr. 2 antigen.

FIG. 4 is a graph showing the titer distribution of antibodies againstpartial WT1 antigens, i.e., the titer distribution of antibodies againstpartial WT1 antigens in healthy subjects and cancer patients, wherein Nand C respectively show the distribution in 54 healthy subjects and thedistribution in 20 cancer patients against the Fr. 1 antigen (a), theFr. 2 antigen (b), and the Fr. 3 antigen (c).

FIG. 5 is a graph showing the results of an antigen inhibition testagainst the Fr. 3 antigen, i.e., the results that the reactivity of theantibody to Fr. 3 is inhibited by Fr. 1 and Fr. 2, wherein C-19 is apolyclonal antibody against the Fr. 3 antigen, No. 3, No. 7, and No. 9are patient serum showing low titers of antibodies against Fr. 1 and Fr.2 and high titers of antibody against Fr. 3, the white bars show theresults in the presence of the Fr. 1 antigen, and the black bars showthe results in the presence of the Fr. 2 antigen.

FIG. 6 includes graphs showing changes in the titer of IgG antibodyagainst each antigen, wherein the black bars show changes in titer ofthe antibody against Fr. 1, the white bars show changes in titer of theantibody against Fr. 2, the horizontal line bars show changes in titerof the antibody against Fr. 3, the oblique line bars show changes intiter of the antibody against the full-length WT1 antigen, and the blackcircles show variations in tumor size measured by MRI.

FIG. 7 includes graphs showing the results of comparison of IgG antibodytiter before vaccination.

FIG. 8 includes graphs showing the results of comparison of IgG antibodytiter after vaccination.

FIG. 9 includes graphs showing IgM antibody titers before vaccination,wherein A shows titers of IgM antibody against Fr. 2, and B shows thetiters of IgM antibody against Fr. 3.

FIG. 10 includes graphs showing IgM and IgG antibody titers beforevaccination.

FIG. 11 is a graph showing IgM and IgG antibody titers in brain tumorpatients, wherein the vertical axis shows IgM antibody titers, thehorizontal axis shows IgG antibody titers, the open circles show theresults of SD patients, the black circles show the results of PDpatients, and the dotted line shows tentative reference values.

FIG. 12 is a graph showing IgM and IgG antibody titers in colon cancerpatients, wherein the vertical axis shows IgM antibody titers, thehorizontal axis shows IgG antibody titers, the open circles show theresults of SD patients, the black circles show the results of PDpatients, and the dotted line shows tentative reference values.

DESCRIPTION OF THE EMBODIMENTS

In the present invention, the anti-WT1 antibody refers to antibodiesagainst a gene product of a zinc finger transcription factor WT1isolated as a responsible gene for Wilms tumor (Wilms tumor gene),specifically, a human WT1 protein (SEQ ID NO: 1) consisting of 449 aminoacids. Such antibodies include a variety of immunoglobulins such as IgGantibody, IgA antibody, and IgM antibody, and these antibodies are allencompassed in the present invention.

In vivo proteins are primarily not recognized as foreign substances bythe immune system and therefore do not usually induce production ofantibodies. The present inventors presumed that an autoantibody to anantigen is produced when the antigen recognized by the autoantibody isconformationally modified to expose an epitope site to the surface andis thereby recognized as a foreign substance. Consequently, it isbelieved that in order to prepare an antigen to be recognized by anautoantibody, an epitope site hidden in the molecule is required to beexposed to the surface by modifying the protein through, for example,genetic engineering for expressing a protein having partial deletion ofthe surface site, cleavage of peptide bond with a protease, treatmentwith an acid, an alkali, or a surfactant, thermal denaturation, ortreatment with a chaotropic reagent such as urea or guanidinehydrochloride.

The present inventors, as shown in examples below, have investigatedmolecules of the WT1 protein that can be an antigen recognized by anautoantibody and have found that the major epitopes are the centralregion (amino acid numbers: 181-324) and the C-terminal region (aminoacid numbers: 294-449) of the amino acid sequence (SEQ ID NO: 1)constituting the human WT1 protein and that use of a peptide at theC-terminal region as an antibody-detecting antigen can particularlyincrease the antibody detection sensitivity to be higher than that whena full-length WT1 antigen is used (Example 1) and that the reactivity ofantibody with the C-terminal side region is inhibited by antigens of theN-terminal side region (amino acid numbers: 1-182) and the centralregion (Example 2) and have proved the above-mentioned points.

The polypeptide having antigenicity to an anti-WT1 antibody used in themethod for measuring the anti-WT1 antibody of the present invention isselected from a polypeptide comprising the amino acid sequence ofpositions 294-449 in SEQ ID NO: 1, a polypeptide comprising the aminoacid sequence of positions 181-324 in SEQ ID NO: 1, a polypeptideincluding an amino acid sequence having deletion, substitution, oraddition of one to several amino acids in the amino acid sequenceconstituting each of these polypeptides, and partial polypeptides ofthese polypeptides.

The polypeptide comprising the amino acid sequence of positions 294-449in SEQ ID NO: 1 corresponds to a zinc finger domain, which is aDNA-binding domain, in the WT1 protein and is also referred to as aC-terminal region in the present invention.

The polypeptide comprising the amino acid sequence of positions 181-324in SEQ ID NO: 1 corresponds to a region between the N-terminal sideregion (amino acid numbers: 1-182) and the C-terminal region in the WT1protein and is also referred to as a central region.

The antigen polypeptide is preferably the polypeptide comprising theamino acid sequence of positions 294-449 or the polypeptide comprisingthe amino acid sequence of positions 181-324 in SEQ ID NO: 1, but may bea partial polypeptide of the polypeptides or a polypeptide including anamino acid sequence having deletion, substitution, or addition of one toseveral amino acids in the amino acid sequence constituting each ofthese polypeptides as long as the antigen polypeptide has antigenicityto an anti-WT1 antibody and can detect the anti-WT1 antibody.

Examples of the partial polypeptide of the polypeptides include peptideseach comprising 6 to 8, preferably 10 to 20, consecutive amino acids ofthe amino acid sequence of positions 294-449 or the amino acid sequenceof positions 181-324 in SEQ ID NO: 1. Specific examples of the partialpolypeptide include a polypeptide comprising the amino acid sequence ofpositions 294-449 in SEQ ID NO: 1, a polypeptide comprising the aminoacid sequence of positions 348-449 in SEQ ID NO: 1, and a polypeptidecomprising the amino acid sequence of positions 181-324 in SEQ ID NO: 1.

The polypeptide comprising the amino acid sequence of positions 294-449and the polypeptide comprising the amino acid sequence of positions181-324 of SEQ ID NO: 1 and partial polypeptides thereof may each havedeletion, substitution, or addition of one to several amino acids in theamino acid sequence thereof as long as the polypeptide has antigenicityto an anti-WT1 antibody.

Throughout the specification, the number of amino acids that can bedeleted, added, or substituted is one or more and is not particularlylimited and is a number of amino acids that can be deleted, added, orsubstituted by a known method such as site-specific mutagenesis and is,for example, one to several tens, preferably one to twenty, morepreferably one to ten, and even more preferably one to five.

Throughout the specification, deletion, addition, or substitution of oneor more amino acid residues in an amino acid sequence means that one ormore amino acid residues are deleted, added, or substituted in arbitraryone or more positions in the same amino acid sequence. The deletion,addition, or substitution may simultaneously occur, and the amino acidresidues that are deleted, added, or substituted may be natural ornon-natural amino acids.

The term “antigenicity to an anti-WT1 antibody” refers to antigenicityto an antibody recognizing a WT1 protein. Since the autoantibodies inWT1-associated disease patients are polyclonal antibodies, the antigenpolypeptide itself of the present invention may have a plurality ofreaction sites (epitopes).

The polypeptide used in the method of the present invention is at leastone polypeptide selected from the above-mentioned polypeptides. In orderto enhance the specificity and measurement sensitivity, a combination ofa plurality of polypeptides may be used.

These polypeptides can be produced by a known genetic engineering usinga WT1 gene, for example, the method described in the example below or amethod in accordance with that or can also be produced by chemicalsynthesis.

The production of the polypeptide by genetic engineering using a WT1gene can be performed by usual genetic recombination conventionallyknown. More specifically, a recombinant DNA capable of expressing adesired WT1 gene in a host cell is prepared, the recombinant DNA isintroduced into host cells for transformation, and the transformant iscultured. The transformant can produce a desired polypeptideintracellularly or extracellularly as an expression product of thetransformant.

Each operation employed here, for example, chemical synthesis of partialgenes, enzyme treatment for cleavage, deletion, addition, or bondingthereof, isolation, purification, selection, and other treatmentthereof, introduction of a recombinant DNA into a host cell, and cultureof the transformant can be performed in accordance with usual methods(see, for example, “Bunshi Idengaku Jikken-ho (Molecular GeneticsExperimental Method)”, Kyoritsu Shuppan Co., Ltd., published in 1993;“PCR Technology”, Takara Shuzo Co., Ltd., published in 1990; Science,224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc.Natl. Acad. Sci. USA., 80, 5990 (1983); Molecular Cloning, by T.Maniatis et al., Cold Spring Harbor Laboratory (1982)).

The polypeptide can also be optionally isolated and purified from theexpression product by various separation procedures utilizing thephysical and chemical properties of the polypeptide (see, for example,“Biochemistry Data Book II”, pp. 1175-1259, First edition, Firstprinting, Jun. 23, 1980, published by Tokyo Kagaku Dojin, Co., Ltd.).

The sample in the present invention is a sample derived from aWT1-associated disease patient or a WT1-associated disease patient aftertreatment, and unlimited examples thereof include body fluids such asblood and urine, which are known to generally contain antibodies.

Examples of the WT1-associated disease include various diseases known asWT1-associated diseases, such as leukemia, solid cancers, andmyelodysplastic syndrome and also include WT1-associated diseases thatmay be found in future.

In the method for measuring an anti-WT1 antibody of the presentinvention, measurement of the anti-WT1 antibody (the concentration ofthe antibody) can be performed by immunoassay using the above-mentionedpolypeptide. The immunoassay may be any known immunoassay, and examplesthereof include radioimmunoassay (RIA), enzyme immunoassay (EIA orELISA), fluoroimmunoassay (FIA), indirect fluorescence assay,luminescent immunoassay, physicochemical assays (TIA, LAPIA, and PCIA),and Western blotting. ELISA is preferably used.

ELISA is a method performed by reacting an antibody to an antigenimmobilized to a solid phase, further reacting a secondary antibodylabeled with an enzyme such as peroxidase or alkaline phosphatase to theantibody bound to the antigen, and then measuring the enzyme label by anappropriate process. Examples of the ELISA include a competitive methodand a sandwich method. The sandwich method (solid-phase sandwich method)is particularly preferred.

The solid-phase sandwich method is performed by, for example, asfollows: A polypeptide of the present invention is immobilized to asolid phase, and a sample to be measured is added thereto. As a result,an antigen-antibody reaction occurs between the solid-phase-immobilizedantigen and an antibody in the sample, and thereby an anti-WT1 antibodypresent in the sample binds to the solid-phased antigen. Subsequently,the bound antibody is detected with an antibody detection reagent tomeasure the anti-WT1 antibody present in the sample.

Alternatively, the antibody detection reagent may be immobilized to asolid phase. An objective anti-WT1 antibody present in a sample can bedetected or measured by capturing antibodies in the sample, then addinga polypeptide of the present invention to the reagent so as to bind tothe anti-WT1 antibody among the captured antibodies, and further bindinga labeled specific antibody to the antigen.

Selection of each process and modification thereof in these measuringmethods are well known to those skilled in the art and are notparticularly limited in the present invention, and any method can beemployed (see, for example, “Rinsho Kensa-ho Teiyo (Clinical ExaminationHandbook)”, Kanehara & Co., Ltd., 1995).

For example, the solid phase used in the solid-phase method may be aninsoluble inactive carrier that is usually used widely. Examples of thecarrier include sticks, beads, microplates, and test tubes made ofvarious materials such as glass, cellulose powder, sephadex, sepharose,polystyrene, filter paper, carboxymethyl cellulose, ion exchange resins,dextran, plastic films, plastic tubes, nylon, glass beads, silk,polyamine-methyl vinyl ether-maleic acid copolymers, amino acidcopolymers, and ethylene-maleic acid copolymers.

The immobilization of an antigen or an antibody is not particularlylimited and may be physical bonding or chemical bonding. Typicalexamples of the immobilization include chemical binding methods such ascovalent bonding methods, e.g., diazo methods, peptide methods (acidamide derivative method, carboxyl chloride resin method, carbodiimideresin method, maleic anhydride derivative method, isocyanate derivativemethod, bromocyan activated polysaccharide method, cellulose carbonatederivative method, condensing reagent method, etc.), alkylation method,crosslinking reagent coupling method (using, for example, glutaraldehydeor hexamethylene isocyanate as the crosslinking reagent), and Ugireaction coupling method; ionic binding methods using supports such asion exchange resins; and physical adsorption methods using porous glasssupports such as glass beads.

The labeling reagent in each measurement system is not particularlylimited, and any conventionally known or expected to come into use infuture can be used. Specifically, those usually used in immunoassay canbe used without any limitation, and examples thereof includeradioisotopes; enzymes such as alkaline phosphatase (ALP) and peroxidase(PDX); fluorescent substances such as fluorescein isothiocyanate (FITC)and tetramethylrhodamine isothiocyanate (RITC); and1N-(2,2,6,6-tetramethyl-1-oxyl-4-piperidyl)-5N-(aspartate)-2,4-dinitrobenzene(TOPA).

Examples of the enzyme labeling material for enzyme labeling include, inaddition to those mentioned above, microperoxidase, chymotripsinogen,procarboxypeptidase, glyceroaldehyde-3-phosphate dehydrogenase, amylase,phosphorylase, D-Nase, and P-Nase. Labeling using these labelingmaterials may be performed according to a known method (see, forexample, “Monoclonal antibody”, Tatsuo Iwasaki, et al., KodansyaScientific, 1984; “Enzyme Immunoassay”, 2nd edition, Eiji Ishikawa, etal., Igaku Shoin, 1982).

The enzyme activity can be measured by a known method depending on thetype of the enzyme used. For example, in a case of using peroxidase as alabeling enzyme, ABTSJ (2,2′-azino-bis(3′-ethylbenzthiazoline sulfonicacid) is used as the substrate; in a case of using alkaline phosphatase,p-nitrophenyl phosphate is used as the substrate, and the decompositionof each substrate is measured with, for example, a spectrophotometer(See, for example, “Enzyme Immunoassay”, 2nd edition, Eiji Ishikawa, etal., Igaku Shoin, 1982).

When a radioisotope or fluorescent material is used instead of theenzyme label as a marker, the marker can also be measured by a knownmethod.

In the measurement system, any solvent that is usually used and does notadversely affect the reaction can be used. Specifically, a buffersolution having a pH of about 5 to 9, such as a citrate buffer solution,a phosphate buffer solution, a tris-hydrochloric acid buffer solution,or an acetate buffer solution can be preferably used.

Immune reaction (binding) conditions are not particularly limited, andusual conditions that are used in these assays are employed. In general,a reaction may be performed at a temperature of 45° C. or less,preferably about 4 to 40° C., for about 1 to 40 hours.

Accordingly, in the method using the polypeptide of the presentinvention as an antigen, an anti-WT1 antibody in a WT1-associateddisease patient can be accurately and sensitively measured, and avariation in the anti-WT1 antibody titer can be satisfactorily detected.

The concentrations of the anti-WT1 antibody in WT1-associated diseasepatients are significantly increased compared to those in healthysubjects and are reduced by treatment of the patients. Accordingly, itis possible to determine the presence, treatment progress, and prognosisof a WT1-associated disease using the concentration of the anti-WT1antibody, preferably the change of the concentration with time, as aclinical index. For example, the anti-WT1 antibody disappears when aWT1-associated disease such as leukemia has completely remitted. Themaintenance of the complete remission state can be confirmed byinvestigating the disappearance of this antibody over time.

Detection of an anti-WT1 antibody in a WT1-associated disease patient,i.e., identification of an anti-WT1 antibody positive patient means thathumoral immune response to WT1 is caused in the patient. Accordingly,the detection itself of an anti-WT1 antibody is useful for determiningor diagnosing the immune response ability of the patient in theWT1-associated disease. Patients showing immune response to WT1 may havebetter prognosis, because of the high immune response, compared topatients not showing immune response. Such determination or diagnosiscan be an index for determining the prognosis of a WT1-associateddisease.

Accordingly, the method for measuring an anti-WT1 antibody of thepresent invention is useful for determining the presence, treatmentprogress, and prognosis of a WT1-associated disease, in particular, forexamination or diagnosis of immune response ability (to cancer) ofvarious cancer patients.

The method for measuring an anti-WT1 antibody of the present inventioncan be applied to prediction of a responder to WT1 vaccine therapy ofcancer before the therapy or to monitoring of response to the therapyafter the therapy. For example, a patient of cancer, such as brain tumoror colon cancer, having a high titer of IgG antibody or of IgM antibodyagainst a peptide in the C-terminal side region (e.g., a polypeptidecomprising the amino acid sequence of positions 294-449 in SEQ ID NO: 1)or a high titer of IgM antibody against a peptide in a central region(e.g., a polypeptide comprising the amino acid sequence of positions181-324 in SEQ ID NO: 1) before administration of a WT1 vaccine can beexpected to obtain high therapeutic effect by the WT1 vaccine.Furthermore, in a responder, the titer of IgG antibody to the peptide ofthe central region increases after WT1 vaccine administration, and atendency of a slight increase in the titer of IgG antibody to thepeptide of the C-terminal side region is also recognized. These resultsdemonstrate that therapeutic response can be monitored using the changein the titer of IgG antibody to the peptide of the central region or theC-terminal side region as an index.

The method for measuring an anti-WT1 antibody of the present inventionor various tests using the method can be easily performed by using thepolypeptide of the present invention as a measurement reagent. Thepresent invention also provides such a measurement reagent. Themeasurement reagent can be used as a test reagent (kit) for determiningthe presence, treatment progress, and prognosis of a WT1-associateddisease.

The measurement reagent or the test reagent of the present inventioncontains the antigen polypeptide of the present invention as an activeingredient. The measurement reagent or the test reagent may furthercontain arbitrary reagents such as an antibody detection reagent used inthe measurement system and reagents necessary for performing themeasurement, e.g., an antibody dilution solution, a reaction dilutionsolution, a buffer solution, a washing solution, and a marker detectionreagent.

EXAMPLES

Examples of the present invention will now be described, but the presentinvention is not limited thereto.

Example 1 1. Material and Method 1-1. Serum Sample

Twenty serum samples of cancer patients collected at Osaka Universityand commercially available 54 serum samples of healthy subjects wereused. Serum of patients treated with a WT1 vaccine was collected beforethe administration and at several points of time after theadministration at Osaka University.

1-2. Measurement of Titer of Antibody to Full-Length WT1 Antigen

The titer of antibody to a full-length WT1 antigen of cancer patientserum was measured by ELISA established by Oji, et al. (Non PatentDocument 7: Oji Y, Kitamura Y, Kamino K, et al., WT1 IgG antibody forearly detection of nonsmall cell lung cancer and as its prognosticfactor, Int J Cancer 2009, 125: 381-7).

1-3. Construction of Partial WT1 Antigen Expression Vector

Full-length WT1 cDNA was divided into an N-terminal region of positions1-182 (Fr. 1), a central region of positions 181-324 (Fr. 2), and aC-terminal region of positions 294-449 (Fr. 3) of WT1 (SEQ ID NO: 1).These regions were amplified by PCR using Fr. 1 amplification primers(SEQ ID NO: 2 and SEQ ID NO: 3), Fr. 2 amplification primers (SEQ ID NO:4 and SEQ ID NO: 5), and Fr. 3 amplification primers (SEQ ID NO: 6 andSEQ ID NO: 7). Fr. 1 and Fr. 2 were each cloned into an expressionvector pET-42a(+) (Merck KGaA), and Fr. 3 was cloned into an expressionvector pQE-80L (QIAGEN N.V.).

SEQ ID NO: 2:  ATGCGCGGTACCATGGGCTCCGACGTGCGGGACCTG SEQ ID NO: 3: ATGCGCGCGGCCGCCATGGGATCCTCATGCTTGAAT SEQ ID NO: 4: ATGCGCGGTACCCCCATGGGCCAGCAGGGCTCGC SEQ ID NO: 5: ATGCGCGCGGCCGCCATGAAGGGGCGTTTCTCACTGG SEQ ID NO: 6: ATGCGCGGATCCTTCAGAGGCATTCAGGATGTGC SEQ ID NO: 7: ATGCGCAAGCTTCAAAGCGCCAGCTGGAGTTTGGTC

1-4. Production of Fr. 1 Antigen

The Fr. 1 expression vector was transformed into E. coli BL21 (DE3), andexpression of recombinant hWT1 Fr. 1 was induced in 1 mM IPTG at 16° C.for 16 hours. The cells were collected by centrifugation, then suspendedin D-PBS(−) containing 0.2% Triton X-100, and sonicated, followed bycentrifugation to collect the soluble fraction. The soluble fraction wasdiluted two-fold with D-PBS(−), bound to a GST fusion proteinpurification column (Glutathione Sepharose HP, GE Healthcare, Inc.)equilibrated with an equilibration buffer solution B1-1 (D-PBS(−)containing 0.1% Triton X-100), washed with the equilibration buffersolution, and then eluted with an elution buffer solution B1-1 (50 mMTris-HCl, 0.2% Triton X-1001, 10 mM reduced glutathione, pH 8.0). Theeluate was further diluted three-fold with an equilibration buffersolution B1-2 (20 mM NaPi, 0.5 M NaCl, 0.2% Triton X-100, pH 7.4), boundto a His-tag purification column (Ni Sepharose HP, GE Healthcare, Inc.)equilibrated with the equilibration buffer solution B1-2, washed withthe equilibration buffer solution B1-2 and a washing buffer solutionB1-2 (20 mM NaPi, 0.5 M NaCl, 0.2% Triton X-100, 250 mM Imidazole, pH7.4) sequentially, and then eluted with an elution buffer solution 1-2(20 mM NaPi, 0.5 M NaCl, 0.2% Triton X-100, 500 mM Imidazole, pH 7.4).The purified Fr. 1 antigen was subjected to protein determination by aBradford method.

1-5. Preparation of Fr. 2 Antigen

The hWT1 Fr. 2 expression vector was transformed into E. coli BL21(DE3), and expression of a Fr. 2 antigen was induced in 1 mM IPTG at 37°C. for 3 hours. The cells were collected by centrifugation, thensuspended in D-PBS(−) containing 0.2% Triton X-100, and sonicated,followed by centrifugation to collect the insoluble fraction. Theinsoluble fraction was further suspended in D-PBS(−) and was collectedby centrifugation. This procedure was repeated twice for washing. Thewashed insoluble fraction was suspended in D-PBS(−) containing 2 M ureaand was incubated at 4° C. for 16 hours. The insoluble fraction wascollected by centrifugation and was further suspended in D-PBS(−)containing 6 M urea, followed by incubation at 4° C. for 16 hours toobtain a soluble fraction. The soluble fraction was diluted three-foldwith an equilibration buffer solution B2 (20 mM NaPi, 0.5 M NaCl, 6 Murea, 5 mM 2-mercaptoethanol, pH 7.4), bound to a His-tag purificationcolumn (Ni Sepharose HP, GE Healthcare, Inc.) equilibrated with anequilibration buffer solution B2, washed with the equilibration buffersolution B2 and a washing buffer solution B2 (20 mM NaPi, 0.5 M NaCl, 6M urea, 5 mM 2-mercaptoethanol, 200 mM Imidazole, pH 7.4) sequentially,and then eluted with an elution buffer solution B2 (20 mM NaPi, 0.5 MNaCl, 6 M urea, 5 mM 2-mercaptoethanol, 500 mM Imidazole, pH 7.4). Thepurified Fr. 2 antigen was subjected to protein determination by aBradford method.

1-6. Preparation of Fr. 3 Antigen

The Fr. 3 expression vector was transformed into E. coli BL21 (DE3), andexpression of a Fr. 3 antigen was induced in 1 mM IPTG at 16° C. for 16hours. The cells were collected by centrifugation, then suspended inD-PBS(−) containing 30 μM ZnCl₂ and 0.2% Triton X-100, and sonicated,followed by centrifugation to collect the soluble fraction. The solublefraction was diluted two-fold with an equilibration buffer solution B5(20 mM Tris-HCl, 1 M NaCl, 0.1% Triton X-100, 30 μM ZnCl₂, pH 8.0),bound to a His-tag purification column (TALON superflow, Clontech, Inc.)equilibrated with the equilibration buffer solution, washed with theequilibration buffer solution 5 and a washing buffer solution B5 (10 mMTris-HCl, 1 M NaCl, 30 μM ZnCl₂, 0.1% Triton X-100, 25 mM Imidazole, pH8.0) sequentially, and then eluted with an elution buffer solution B (10mM Tris-HCl, 0.72 M NaCl, 30 μM ZnCl₂, 0.1% Triton X-100, 200 mMImidazole, pH 8.0). The purified Fr. 3 antigen was subjected to proteindetermination by a Bradford method.

1-7. Production of Antigen-Immobilized Solid-Phase Plate

The purified Fr. 1 antigen, Fr. 2 antigen, or Fr. 3 antigen was adjustedto a concentration of 10 μg/mL with D-PBS(−), and 100 μL thereof wasadded to a 96-well microtiter plate (96-Well EIA/RIA Stripwel Plate,CORNING, Inc.), followed by incubation at 4° C. for 16 hours forimmobilization to a solid phase.

The plate was washed with a washing solution (D-PBS(−) containing 0.05%Tween 20) once, and 300 μL of a plate blocking solution (D-PBS(−)containing 1% bovine serum albumin (BSA)) was added to the plate,followed by incubation at 4° C. for 16 hours for blocking. The blockingsolution was removed, and the plate was dried in a 25° C. incubator andwas then stored at 4° C. until use.

1-8. Measurement of IgG by ELISA Using Fr. 1 Antigen

An antigen-immobilized solid-phase plate was washed with the washingsolution once, and 100 μL of a commercially available antibody (hWT1H-290, SANTA CRUZ Biotechnology, Inc.) or serum appropriately seriallydiluted with a sample dilution solution 1 (20 mM NaPi, 0.65 M NaCl,0.05% Tween 20, 0.05% ProClin 300, 1% BSA, pH 8.0) was added to theantigen-immobilized solid-phase plate, followed by a reaction withshaking at 25° C. for 1 hour. Subsequently, the plate was washed withthe washing solution three times, and 100 μL of horse-radish peroxidase(HRP)-conjugated Protein G (Acris Antibodies Inc.) diluted 50000-foldwith a secondary reaction dilution solution (D-PBS(−) containing 0.05%Tween 20, 0.05% ProClin 300, and 0.5% BSA) was added to the plate,followed by a reaction with shaking at 25° C. for 1 hour. Finally, theplate was washed with the washing solution three times, and 100 μL of3,3′,5,5′-tetramethylbenzidine (TMB) was added to the plate for coloringat room temperature for 10 minutes. The reaction was stopped with 100 μLof 1 N sulfuric acid. The absorbance at 450 nm (reference wavelength:650 nm) was measured with a microplate reader.

1-9. Measurement of IgG by ELISA Using Fr. 2 Antigen

A GST-expressing E. coli extract was added to a commercially availableantibody (hWT1 H-290) or serum appropriately serially diluted with thesample dilution solution 1 to give a concentration of 5 μg/mL, and themixture was subjected to a reaction with shaking at 25° C. for 1 hour toobtain a Fr. 2 sample solution. An antigen-immobilized solid-phase platewas washed with the washing solution once, and 100 μL of the Fr. 2sample solution was added to the plate, followed by a reaction withshaking at 25° C. for 1 hour. Subsequently, the plate was washed withthe washing solution three times, and 100 μL of HRP-conjugated Protein Gdiluted 50000-fold with the secondary reaction dilution solution wasadded to the plate, followed by a reaction with shaking at 25° C. for 1hour. Finally, the plate was washed with the washing solution threetimes, and 100 μL of TMB was added to the plate for coloring at roomtemperature for 10 minutes. The reaction was stopped with 100 μL of 1 Nsulfuric acid. The absorbance at 450 nm (reference wavelength: 650 nm)was measured with a microplate reader.

1-10. Measurement of IgG by ELISA Using Fr. 3 Antigen

An antigen-immobilized solid-phase plate was washed with the washingsolution once, and 100 μL of a commercially available antibody (hWT1H-290) or serum appropriately serially diluted with a sample dilutionsolution 2 (20 mM NaPi, 0.05% Tween 20, 0.05% ProClin 300, 1% BSA, pH8.0) was added to the plate, followed by a reaction with shaking at 25°C. for 1 hour. Subsequently, the plate was washed with the washingsolution three times, and 100 μL of HRP-conjugated Protein G diluted50000-fold with the secondary reaction dilution solution was added tothe plate, followed by a reaction with shaking at 25° C. for 1 hour.Finally, the plate was washed with the washing solution three times, and100 μL of TMB was added to the plate for coloring at room temperaturefor 10 minutes. The reaction was stopped with 100 μL of 1 N sulfuricacid. The absorbance at 450 nm (reference wavelength: 650 nm) wasmeasured with a microplate reader.

1-11. Measurement of IgM by ELISA Using Each Antigen

IgM antibody was measured as in the measurement of IgG antibody. Thedetection was performed through a reaction of a 50000-fold dilutedHRP-conjugated anti-human IgM specific antibody.

2. Results 2-1. Titer of Antibody to Full-Length WT1 Antigen

Table 1 and FIG. 1 show the blood anti-WT1 antibody titers of cancerpatient serum samples measured by the method of Oji, et al. According tothe report by Oji, et al., the titer distribution of antibodies againstthe full-length WT1 antigen in healthy subjects is 10 to 3664 WRU, andthe median thereof is 392 WRU (Non Patent Document 7). The titerdistribution of antibodies in 20 cancer patient serum samples used inthis test was 7 to 1682 WRU. Thus, all the samples were within the rangeof healthy subjects.

TABLE 1 WT1 antibody Fr. 1 antibody Fr. 2 antibody Fr. 3 antibody titertiter titer titer No. (WRU) (unit) (unit) (unit) 1 102 1.9 54.4 367.9 242 4.5 22.2 258.5 3 103 5.2 74.4 184.7 4 103 3.7 78.7 485.2 5 14 4.0 9.473.8 6 968 8.7 134.5 5045.1 7 242 9.2 49.8 253.4 8 1143 8.6 154.4 5391.59 242 21.0 43.2 580.2 10 1682 29.9 229.9 9087.8 11 453 7.1 69.9 1029.612 237 5.2 42.2 337.8 13 499 6.2 56.2 559.9 14 1389 21.2 166.9 7887.0 15984 19.3 204.9 8326.8 16 7 1.6 10.0 47.5 17 14 2.8 14.9 115.2 18 152411.8 150.4 7826.5 19 42 2.1 10.0 208.6 20 306 6.8 104.0 784.7

2-2. Titer of Antibody to Partial WT1 Antigen in Cancer Patient

Blood antibody titers were measured by ELISA using partial WT1 antigens.The blood antibody titers were calculated from a calibration curve ofabsorbance versus concentration constructed using a serially dilutedcommercially available antibody as a standard. The titers of antibodyagainst each antigen in blood of cancer patients are shown in Table 1.

FIG. 2 shows the results of comparison between the titers of antibodiesagainst each partial WT1 antigen and the full-length WT1 antigen. Thetiters of antibodies against the Fr. 2 antigen and the Fr. 3 antigenshowed high correlations with the titer of antibody against thefull-length WT1 antigen. The correlation between the titer of antibodyagainst the Fr. 2 antigen and the titer of antibody against the Fr. 3antigen was also high (FIG. 3).

These results suggest that the titer of antibody against the full-lengthWT1 antigen reflects the titer of antibody against Fr. 3 and that Fr. 3includes a major epitope to the antibody in blood.

2-3. Titer Distribution of Antibody Against Partial WT1 Antigen

FIG. 4 shows the results of comparison between the titer distribution ofantibodies against partial WT1 antigens in 54 healthy subjects (N) andthe titer distribution of antibodies against partial WT1 antigens in 20cancer patients (C). The titer distributions of antibodies against theFr. 2 antigen and the Fr. 3 antigen in the cancer patients were higherthan those in the healthy subjects, whereas the titer distributions ofantibodies against the Fr. 1 antigen were substantially the same in thecancer patients and the healthy subjects. In particular, the titerdistribution of antibodies against the Fr. 3 antigen was most broad, andthe antibody titers of six cancer patient samples were five or moretimes higher than those of the healthy subjects.

The results above demonstrate that the Fr. 3 antigen can classify a hightiter group more clearly than the case using the titer of antibodyagainst the full-length WT1 antigen.

Example 2 Antigen Inhibition Test

In order to investigate the phenomenon that the titer of antibodyagainst the Fr. 3 antigen is detected with higher sensitivity comparedto the titer of antibody against the full-length WT1 antigen, whetherthe antibody epitope of the Fr. 3 antigen is masked by reacting the Fr.1 antigen or Fr. 2 antigen to the Fr. 3 antigen was investigated. As thesamples, a polyclonal antibody (PoAb) against Fr. 3 and the serum ofthree patients (sample Nos. 3, 7, and 9) having high titers of antibodyagainst Fr. 3 were used.

1. Method

A Fr. 3 antigen-immobilized solid-phase plate was washed with thewashing solution once, and 100 μL of the Fr. 1 antigen or Fr. 2 antigendiluted with the secondary reaction dilution solution was added to theplate, followed by a reaction with shaking at 25° C. for 1 hour.Subsequently, 100 μL of a commercially available antibody (hWT1 C-19,SANTA CRUZ Biotechnology, Inc.) or serum diluted with the sampledilution solution 1 was added to the plate, followed by a reaction withshaking at 25° C. for 1 hour. Furthermore, the plate was washed with thewashing solution three times, and then 100 μL of HRP-conjugated ProteinG diluted 50000-fold with the secondary reaction dilution solution wasadded to the plate, followed by a reaction with shaking at 25° C. for 1hour. Finally, the plate was washed with the washing solution threetimes, and 100 μL of TMB was added to the plate for coloring at roomtemperature for 10 minutes. The reaction was stopped with 100 μL of 1 Nsulfuric acid. The absorbance at 450 nm (reference wavelength: 650 nm)was measured with a microplate reader, and the reaction inhibition rate(B/BO) was calculated assuming that the value in the absence of theantigen was 100%.

2. Results

The results are shown in FIG. 5.

Fr. 1 and Fr. 2 inhibited the reactivity of PoAb to Fr. 3 by 25% and50%, respectively. Similarly, Fr. 1 or Fr. 2 inhibited the antibodytiter of each serum sample to Fr. 3. In sample No. 3, the reaction wasnot inhibited by Fr. 1, but 70% thereof was inhibited by Fr. 2. Insample No. 9, the reaction was not inhibited by Fr. 2, but 40% thereofwas inhibited by Fr. 1. In sample No. 7, though the degrees were low,the reaction was inhibited by both Fr. 1 and Fr. 2 by 10% and 20%,respectively.

The results above suggest that Fr. 1 and Fr. 2 have capability ofbinding to Fr. 3 and that the binding masks the antibody epitope of Fr.3.

Example 3 Evaluation of Antibody Titer in Brain Tumor PatientAdministered with WT1 Vaccine

The titers of IgG and IgM antibodies against the Fr. 1 antigen, Fr. 2antigen, and Fr. 3 antigen of patients administered with a modified WT1235 peptide vaccine (amino acid sequence (YTWNQMNL)) were evaluated. Asthe patient samples, five samples of the stable disease (SD) group andfour samples of the progression disease (PD) group were used. The cancertissues were evaluated by MRI.

FIG. 6 summarizes the changes in IgG antibody titer of patientsadministered with the WT1 235 peptide.

The titer of IgG antibody against Fr. 1 was hardly detected in allpatients. The titers of antibody against Fr. 3 were obviously classifiedinto a high value group and a low value group and hardly changed beforeand after the administration. In addition, these antibody titerssubstantially did not show a correlation with the full-length WT1antigen. However, the titer of antibody against Fr. 2 increased withtime after vaccination in some patients. This antibody titer wasconfirmed to have a high correlation with the titer of antibody againstthe full-length WT1 antigen.

FIG. 7 summarizes the results of comparison of the titer of IgG antibodyagainst each antigen before vaccination and therapeutic response.

The results show that the titers of antibody against Fr. 3 were 100units or more in four of five samples of the SD group and that theantibody titers were less than 100 units in all four samples of the PDgroup. Such tendencies were not recognized in the titers of antibodiesagainst other antigens. The results above show that patients having hightiters of IgG antibody against Fr. 3 before vaccination are patients whorespond to the vaccine therapy.

FIG. 8 summarizes the results of comparison of the titer of IgG antibodyagainst each antigen after vaccination and therapeutic response. Theresults show that the titers of antibodies against Fr. 2 and thefull-length WT1 antigen at three months after vaccination were two-foldor more higher than those before vaccination in three of five samples ofthe SD group, whereas the antibody titers in all four samples of the PDgroup hardly increased. The results above show that patients havingincreased titers of IgG antibody against Fr. 2 after vaccination arepatients who respond to the vaccine therapy.

The titers of IgM antibodies against Fr. 2 and Fr. 3 were evaluatedusing samples before vaccination. FIG. 9 summarizes the results ofcomparison of IgM antibody titer and therapeutic response. The resultsshow that the titers of antibody against Fr. 2 were 200 units or more inall five samples of the SD group and that the antibody titers were lessthan 200 units in all four samples of the PD group. In addition, thetiters of antibody against Fr. 3 were 80 units or more in three of fivesamples of the SD group, and the antibody titers were less than 80 unitsin all four samples of the PD group. The results above show thatpatients having high titers of IgM antibodies against Fr. 2 and Fr. 3before vaccination are patients who respond to the vaccine therapy.

Example 4 Evaluation of Antibody Titer in Colon Cancer PatientAdministered with WT1 Vaccine

The titers of IgG and IgM antibodies against each antigen in patientsadministered the WT1 235 peptide were evaluated using eight samples ofthe SD group and 14 samples of the PD group as patient samples. Thetiters of antibodies against Fr. 2 and Fr. 3 were evaluated usingsamples before the vaccination. The results are summarized in FIG. 10.

The titers of IgG and IgM antibodies against Fr. 3 in the SD group wereobviously higher than those in the PD group. The titers of antibodyagainst Fr. 3 were 140 units or more in four of eight samples of the SDgroup, whereas the antibody titer was 140 units or less in all 14samples of the PD group. The titers of IgM antibody against Fr. 3 were140 units or more in six of eight samples of the SD group, whereas theantibody titers were 140 units or less in 10 of 14 samples of the PDgroup. The results above show that patients having high titers of IgMand IgG antibodies against Fr. 3 before vaccination are patients whorespond to the vaccine therapy.

Example 5 Stratification of Responders to WT1 Vaccine Therapy BeforeTherapy

The results of evaluation of the titers of IgM and IgG antibodiesagainst each antigen show that the titer of antibody against Fr. 3before vaccination is associated with the therapeutic response in bothbrain tumor patients and colon cancer patients. Accordingly, the titersof IgM and IgG antibodies against Fr. 3 before vaccination weresummarized in brain tumor patients and in colon cancer patientsseparately. The results of brain tumor patients are shown in FIG. 11.

In the brain tumor patients, when 100 units are prescribed as referencevalues of the IgG antibody titer and the IgM antibody titer, either IgMor IgG antibody titer was equal to or higher than the reference value inall samples of the SD group, whereas both IgM and IgG antibody titerswere equal to or lower than the reference values in all samples of thePD group, which allows stratification. That is, the sensitivity andspecificity in selection of responders before vaccination are both 100%.The results of colon cancer patients are shown in FIG. 12. When 150units are prescribed as reference values of the IgG antibody titer andthe IgM antibody titer, either IgM or IgG antibody titer was equal to orhigher than the reference value in seven of eight samples of the SDgroup, whereas both IgM and IgG antibody titers were equal to or lowerthan the reference values in 10 of 14 samples of the PD group, whichallows stratification. That is, the sensitivity and specificity inselection of responders before vaccination are 85% and 71%,respectively.

The results above demonstrate that stratification before therapy ofresponders to the therapy is possible even in different cancer types bymeasuring the titers of IgM and IgG antibodies against Fr. 3 beforevaccination.

1. A method for measuring an anti-WT1 antibody in a sample, the methodcomprising: contacting a sample with at least one polypeptide havingantigenicity to the anti-WT1 antibody selected from the group consistingof i) a polypeptide consisting of an amino acid sequence of positions181-324 in SEQ ID NO: 1, and ii) a polypeptide consisting of an aminoacid sequence where the amino acid sequence of i) the polypeptide hasbeen subjected to at least one of deletion, substitution, and additionof 1 to 5 amino acids; and measuring a concentration of the anti-WT1antibody in the sample.
 2. The method according to claim 1, wherein thecontacting comprises immobilizing at least one of i) the polypeptide andii) the polypeptide to a solid phase and contacting at least oneimmobilized polypeptide with the sample, and the measuring comprisesdetecting a reaction product between at least one immobilizedpolypeptide and an anti-WT1 antibody present in the sample to measure aconcentration of the anti-WT1 antibody.
 3. A method for diagnosing aWT1-associated disease, comprising: contacting a sample from a subjectwith at least one polypeptide having antigenicity to an anti-WT1antibody selected from the group consisting of i) a polypeptideconsisting of an amino acid sequence of positions 181-324 in SEQ ID NO:1, and ii) a polypeptide consisting of an amino acid sequence where theamino acid sequence of i) the polypeptide has been subjected to at leastone of deletion, substitution, and addition of 1 to 5 amino acids; anddetecting the anti-WT1 antibody in the subject.
 4. The method accordingto claim 3, wherein the WT1-associated disease is leukemia.
 5. A methodfor predicting a responder to or for therapeutic monitoring of WT1vaccine therapy of cancer, comprising: contacting a sample from asubject with at least one polypeptide having antigenicity to an anti-WT1antibody selected from the group consisting of i) a polypeptideconsisting of an amino acid sequence of positions 181-324 in SEQ ID NO:1, and ii) a polypeptide consisting of an amino acid sequence where theamino acid sequence of i) the polypeptide has been subjected to at leastone of deletion, substitution, and addition of 1 to 5 amino acids; anddetecting the anti-WT1 antibody in the subject.
 6. The method accordingto claim 5, wherein the cancer is brain tumor or colon cancer.
 7. Acomposition, comprising: at least one polypeptide having antigenicity tothe anti-WT1 antibody selected from the group consisting of i) apolypeptide consisting of an amino acid sequence of positions 181-324 inSEQ ID NO: 1, and ii) a polypeptide consisting of an amino acid sequencewhere the amino acid sequence of i) the polypeptide has been subjectedto at least one of deletion, substitution, and addition of 1 to 5 aminoacids.
 8. The composition according to claim 7, wherein i) thepolypeptide is included.
 9. A method for detecting an autoantibody,comprising: modifying a WT1 protein such that a modified WT1 protein hasa surface where an epitope site of a polypeptide is exposed; andcontacting a sample from a subject with the modified WT1 protein suchthat an autoantibody that recognizes the modified WT1 as an antigen isdetected, wherein the polypeptide comprises at least one selected fromthe group consisting of i) a polypeptide consisting of an amino acidsequence of positions 181-324 in SEQ ID NO: 1, and ii) a polypeptideconsisting of an amino acid sequence where the amino acid sequence of i)the polypeptide has been subjected to at least one of deletion,substitution, and addition of 1 to 5 amino acids.
 10. The methodaccording to claim 1, wherein the measuring of the concentration of theanti-WT1 antibody comprises performing one of radioimmunoassay, enzymeimmunoassay, fluoroimmunoassay, indirect fluorescence assay, luminescentimmunoassay, physicochemical assay, and Western blotting.
 11. The methodaccording to claim 1, wherein the measuring of the concentration of theanti-WT1 antibody comprises performing ELISA.
 12. The method accordingto claim 1, wherein the contacting comprises contacting the sample withi) the polypeptide consisting of the amino acid sequence of positions181-324 in SEQ ID NO:
 1. 13. The method according to claim 12, whereinthe contacting comprises immobilizing i) the polypeptide to a solidphase to obtain an immobilized polypeptide and contacting the samplewith the immobilized polypeptide, and the measuring comprises detectinga reaction product between the immobilized polypeptide and an anti-WT1antibody present in the sample to measure a concentration of theanti-WT1 antibody.
 14. The method according to claim 12, wherein themeasuring of the concentration of the anti-WT1 antibody comprisesperforming one of radioimmunoassay, enzyme immunoassay,fluoroimmunoassay, indirect fluorescence assay, luminescent immunoassay,physicochemical assay, and Western blotting.
 15. The method according toclaim 12, wherein the measuring of the concentration of the anti-WT1antibody comprises performing ELISA.
 16. The method according to claim3, wherein the contacting comprises contacting the sample with i) thepolypeptide consisting of the amino acid sequence of positions 181-324in SEQ ID NO:
 1. 17. The method according to claim 16, wherein theWT1-associated disease is leukemia.
 18. The method according to claim 3,further comprising: determining the subject's immune response abilitybased on whether the anti-WT1 antibody is detected in the subject. 19.The method according to claim 16, further comprising: determining thesubject's immune response ability based on whether the anti-WT1 antibodyis detected in the subject.
 20. The method according to claim 17,further comprising: determining the subject's immune response abilitybased on whether the anti-WT1 antibody is detected in the subject.